Antibodies are proteins produced by the B-lymphocytes of the immune system in response to antigens, recognize antigens and bind to antigens. Such antibodies are regarded as new protein drug candidates for treating diseases. To find desired functional antibodies, various antibody libraries are constructed, and the functional antibodies are screened from antibody libraries. Such antibody libraries are constructed using gene recombination technology. Specifically, genes encoding antibody proteins are extracted from B-cells in the human body to construct antibody gene libraries, and antibodies having desired antigen binding specificity are screened from the libraries. Antibody library technology brought about a revolution in the construction of antibodies such as human antibodies. The most prominent characteristic of antibody immune responses is that antibody binding specifically to a kind or shape of antigen could be made within one week, if the antigen is a foreign substance different from an in vivo component. Antibodies are produced by B-lymphocytes, and a single B lymphocyte produces only one type of antibody. In fact, it is known that numerous B lymphocytes exist in the human body, and each B lymphocyte expresses an antibody having unique antigen binding specificity on the cell membrane. It is generally known that an antigen binding diversity of about 108 exists in the human body. When an antigen invades the body, only B lymphocytes expressing an antibody that binds specifically to the antigen proliferate rapidly while producing a large amount of the antibody, and as a result, the concentration of the antibody in the serum increases rapidly to thereby quickly eliminate the invaded antigen. Thus, an antibody diversity of several hundred millions exists in the human body, and this antibody diversity is referred to as repertoire. Thus, when a sufficient number of B lymphocytes are collected from the human body by blood collection, after which mRNA is isolated from the cells and synthesized into cDNA encoding the heavy-chain and light-chain variable regions of antibody by RT-PCR (reverse transcriptase-polymerase chain reaction), a human antibody repertoire can be constructed in vitro in the form of genes in a relatively simple manner. The key of antibody library technology is to express (or display) this human antibody gene repertoire as protein while paring a gene encoding the antibody protein through any medium (genotype-phenotype linkage), thereby testing an antibody binding to a specific antigen screened from the antibody library and obtaining a gene encoding the specific antibody. Herein, perfect immunity is not required, the repertoire is either displayed as Fab of an antibody having antigen binding function, or displayed as an antibody fragment, named scFv (single-chain variable fragment) in which the heavy-chain and light-chain variable domains (VH and VL) are connected to each other by a short peptide linker of about 15 amino acids. Herein, the display is classified into phage display, ribosome display, yeast display and the like according to the kind of medium that is used in the genotype-phenotype linkage, and an antibody having desired antigen binding characteristics can be obtained without inducing an immune response by administration of an antigen. However, there are shortcomings in that a lot of know-how is required for antibody library construction and antibody screening, it is not easy to obtain high-affinity antibodies, and thus antibody optimization procedures such as affinity maturation are frequently performed after antibody screening, and functional analysis in mammalian cells cannot be performed due to problems such as toxicity, particularly during first-step screening. Such shortcomings have become a barrier for the development of therapeutic antibodies, because therapeutic antibodies do not simply bind to antigens but should have therapeutic functions.
Among antibody libraries, phage display antibody libraries are currently most frequently used. In fact, Humira (anti-TNF-alpha human monoclonal antibody) which is a currently commercially available rheumatoid arthritis therapeutic agent is a therapeutic antibody made by phage display technology. An ideal antibody library contains enormous antibody diversity, and thus high-affinity antibody clones having desired antigen binding specificity can be screened therefrom. For this purpose, a library having an antibody library of about 1010-1011 should be constructed. However, it is very difficult to construct a library having this size by antibody gene cloning, and this is considered as the most difficult problem in the construction of phage display antibody libraries. In addition, there is a shortcoming in that functional analysis cannot be directly performed, because phages themselves act to be toxic. The biggest advantage of ribosome display technology is a cell-free system, and thus theoretically, libraries having a large size of 1013 can be easily constructed by ribosome display technology. Thus, ribosome display technology is advantageous for the screening of high-affinity antibodies (generally, the size of an antibody library becomes larger, the possibility for high-affinity antibodies to be contained in the library is higher). In addition, because PCR amplification is performed in ribosome display technology, error-prone polymerase or the like can be used, and thus the introduction of mutation for artificially inducing is very easy. However, ribosome display technology also has toxicity problems and various experimental problems. For this reason, phage display technology is mainly used for the construction of antibody libraries of naive origin. In yeast display technology, there are many technical limitations in making antibody libraries having a diversity of 109 or more, because a process of inserting a recombinant vector into a S. cerevisiae strain is required and the size of yeast cells is large. Thus, yeast display technology is mainly used to construct a mutant library of already established antigen-specific antibodies and to screen high-affinity antibodies from the mutant library.
However, in such antibody libraries, all antibodies are not individually separated, but are mixed together. Such antibody libraries have limitations in that screening of an antibody to a target antigen based on its function (activity) is not actually impossible, and only screening of an antibody based on binding to an antibody is possible. Initial antibody candidates obtained in this procedure are examined for their function in a subsequent step to select antibodies having functions. In most cases, antibodies, which easily bind but have no function, are obtained in the selection step. Thus, a new method that overcomes the limitation of this screening method is required. In other words, a method of screening antibodies based on their function from beginning is required. However, existing libraries are in a state in which various antibodies are mixed together, and it is impossible to screen individual antibodies based on its function. Thus, if it is possible to individually purify and store all antibodies in specially addressed library, like low-molecular-weight compound libraries, it is possible to screen antibodies based on their function. However, because antibodies are proteins, processes for expressing and purifying antibodies are required, and thus it is actually impossible to construct a library of 100,000 or 1,000,000 different antibodies. In other words, conventional methods have shortcomings in that, when the library diversity is assumed to be 1,000,000, the purification of 1,000,000 proteins is required, and the number of required protein purifications increases by exponentially as the diversity increases. Conventional library construction technologies include a technology of constructing a library by combining VH and VL at the DNA level in a vector (U.S. Pat. No. 8,178,320), a technology of constructing a library of antibody light chains and heavy chains at the DNA level (U.S. Pat. No. 7,858,559), etc. However, these library construction technologies have shortcomings in that the purification of a desired number of proteins is required to construct a library having a diversity satisfying a combination of the proteins at the DNA level, and the functions of the antibodies in the constructed library cannot be immediately analyzed due to the geometric number of the antibodies, and for this reason, an additional step of reducing the number of antibodies, which can be screened by binding to antigens and analyzed for their function, is required, and a true important antibody can be missed during this screening. Particularly, in conventional library construction methods, Fvs should be expressed in combination at the DNA level, and thus the purification of proteins corresponding to the library diversity is required. Thus, in the conventional library construction methods, it is impossible to construct a library containing individually separated antibodies.
Under such circumstances, the present inventors have made extensive efforts to develop a library in which antibodies are individually separated so that they can be functionally screened. As a result, the present inventors have paid attention to the construction of library, in which combinations happened at the protein level, unlike conventional library construction technologies of combining antibody domains at the DNA level, and have found that an Fv library based on a combination of proteins can be constructed by combining VH and VL at the protein level, thereby completing the present invention.